Iron is required and cannot be replaced by other metals, for many essential aspects of a living cell's physiology and metabolism, whether the cell is a spoilage causing microbe in a product intended for use by humans or a pathogenic cell within the body and capable of causing human or animal or fish disease, i.e., such as a microbial pathogen (bacterial, fungal or parasitic) or a pathogenic animal cancer cell. The only known exception to this essential requirement for iron is with certain non-pathogenic Lactobacilli bacteria.
This generally universal iron requirement could therefore be a useful target for new means to interfere with or a stop the growth of cells. To date, only limited advances have been made in affecting iron nutrition of cells due to a lack of suitable chemical compounds that possess the needed characteristics. Bacterial, fungal, parasitic and animal cells normally possess one or more of various Fe uptake mechanisms that operate at the cell membrane/external environment boundary and these cellular mechanisms essentially serve to internalize Fe from the external environment for use within the cell as shown in FIG. 1.
Iron reduction from an enzymatic surface receptor/reduction/transport system (I) is important for making iron that predominates in aerobic environments as insoluble Fe3+ into the more soluble Fe2+ form, and this mechanism is found in most bacterial, fungal and animal cells. Pathogenic bacteria and yeasts generally possess multiple Fe uptake mechanisms while animal cells do not produce or utilize microbial type siderophores. Siderophores are chelating compounds produced primarily by microbial cells. Rather than using a siderophore, vertebrate animal cells utilize the protein transferrin (II) that is typically produced by liver cells of the animal and which circulates to shuttle iron from the gut through the blood stream and to all other cells of the body. Certain pathogenic microorganisms have developed an ability to bind and utilize transferrin Fe by transferring this to a shuttle carrier in the membrane without taking up the transferrin molecule into the cell (II). Other bacteria and fungi can take up heme, another iron carrying compound produced by microbial and animal cells. Heme can be taken into the cell directly by a receptor/transport system (II) and the cells then use the heme iron internally. Various bacteria and fungi can utilize various heterologous siderophores as produced by other microbes by removing iron from these at the cell surface shuttle system (III). The iron reduction mechanism (I) may potentially play a role in iron removal from heterologous siderophores or transferrin in some cells. Various bacterial and fungal pathogens produce their own autologous siderophores in response to iron need, secrete these into the extracellular environment and then take these back up with iron as chelated from the external environment (IV). Cells of parasitic animals have been studied less but some are known to acquire heme and it is likely that they employ acquisition mechanisms similar to other eukaryotic cells such as the fungi or animal cells. Without wishing to be bound by theory, Table 1 below provides a further comparison of the various iron acquisition mechanisms as diagrammed in FIG. 1 and discussed above.
TABLE 1Summary of Iron Acquisition Mechanisms of CellsType(see diagram)MechanismBacteriaFungiAnimalIEnzymatic FeAssists uptakeFor direct FeDirect uptakereduction andfrom variousuptake and alsoanalogous totransportsiderophoresin conjunctionyeast systemwithsiderophoreuptakeIIReceptor/UptakePathogens such asHeme uptake byNormalfor Heme orStaphylococcuspathogenicuptakebinding ofaureus can accessyeasts such asinvolvesTransferrins forheme andCandidareceptors forstripping of iron for transferrinsalbicanstransferrinuptakeIIIReceptor/UptakeCan be associatedFound inNot presentfor heterologouswith reductionvarious typesbut lowsiderophoressystem; aincludingmolecularcommon shuttlepathogenicweightsystem found inyeasts such aschelatorsvarious bacteriaCandidaenter cellalbicansIVProduction/Release/Common with anPathogenicAnimal cellsUptake ofarray ofCandidause animalAutologoushydroxamate andproduce whiletransferrinsSiderophorescatecholate typesother nonanalogous toproduced bypathogen yeastssiderophoresdifferent bacteriado not
There are variations of the simplified generalized Fe uptake mechanisms as summarized in Table 1 and diagrammed in FIG. 1, as could be found for specific cell species. However, for the purposes of this disclosure, the four generalized mechanisms (I-IV) adequately summarize general Fe nutrition for bacteria, fungi, and animal cells including the cells of man and other animals including parasitic cells. It will be appreciated that there are two unifying features that Fe as needed internally by a cell is either off-loaded from a molecule carrying the Fe after the molecule is intercepted at the cell surface by a receptor/transport system or, the Fe is taken up directly into the cell along with the molecule carrying the iron.
Conventional metal chelating compounds such as the iron chelators deferoxamine (also called desferrioxamine B or Desferal™ as marketed by Novartis Ltd.) or deferiprone (1,2-dimethyl-3-hydroxy-pyrid-4-one, as marketed by Apotex Pharmaceutical Company) are already used for medical purposes related to treating human iron metabolic disorders. For these disorders, these compounds chelate iron in the body and provide for its excretion as a soluble low molecular weight iron-chelator complex. Their use has also been proposed for the treatment of infection and cancer. Thus, U.S. Pat. No. 5,663,201 disclosed the use of desferrioxamine B salts for the treatment of cancer while U.S. Pat. Nos. 5,256,676 and 6,825,204 disclose the use of 3-hydroxy-pyrid-4-ones, such as deferiprone for the treatment of parasitic infections. Additionally, deferiprone or hydroxamates such as desferal have been proposed in U.S. Pat. No. 5,302,598 as adjuncts to antibiotics for the treatment of Pneumocystis carni parasitic infection. Other microbial chelators such as exochelin have been proposed in U.S. Pat. No. 5,837,677 for the treatment of cancer. Various chelators have also been disclosed as adjuncts to antibiotics, preservatives or anti-microbial agents such as those disclosed in U.S. Pat. Nos. 6,793,914; 6,267,979; 5,573,800; 6,165,484 and 6,893,630.
Other N-substituted (U.S. Pat. No. 6,932,960) or cycloalkyl (U.S. Pat. No. 7,410,985) derivatives of 3-hydroxy-4-pyridinones have been described for use as alternate pharmaceuticals to relieve medical conditions of iron overload or to treat parasitic infection or other diseases.
However, all these previously disclosed low molecular weight chelators as mentioned above, (i.e., chelators having a low molecular weight of for example 1500 Daltons or less) suffer from a common problem. The problem is that most cells including pathogenic cells can readily access and use these low molecular weight chelators as sources for their needed iron. Molecules of a size of around 1500 Daltons or less can permeate the cellular membrane of prokaryotic (e.g., bacteria) and eukaryotic (e.g., fungal and animal) cells. Thus, the iron chelates of these conventional low molecular weight compounds and compositions of low molecular weight are potentially exploitable for iron by certain bacterial and other cells that would be desirable to control, i.e., through the use by such cells of one of the iron acquisition mechanisms shown in FIG. 1. This fundamental problem severely limits the potential use of the previously disclosed chelators and compositions for controlling cell growth.
Moreover, inappropriate use of one of these previously disclosed chelators in attempt to control a cell that can utilize the particular chelator employed may be a problem and potentially worsen the situation of preservation, infection control or cancer control. In this regard, deferiprone and similar chelators, such as those disclosed in U.S. Pat. No. 6,767,741, are known to provide iron for animal cells in laboratory culture. Therefore, these chelators can not be expected to be useful for treating animal cancer cells. Citric acid is an example of a chelator that meets the definition of a suitable chelator as was disclosed in U.S. Pat. Nos. 6,165,484 and 6,267,979 but citrate is often used to make iron soluble and available in culture media that is used to grow a variety of cells (Porterfield, J., S. 1978) Similarly, chelators, such as ethylene-diamine-tetra-acetic acid (EDTA) as disclosed in U.S. Pat. No. 6,767,741 for controlling growth of cells is used to supply metals in growth media for plants and other cells (Hughes and Poole. 1989). Thus, known soluble chelators of a low molecular weight of less than approximately 1500 Daltons such as EDTA and the entire chemical family of its related compounds may be problematic as chelators for use in the control of infection, cancer or microbial spoilage, given that many cells can potentially utilize these for iron delivery.
Gram negative bacteria comprise a principal category of infection causing bacteria and these have been shown to possess a generalized Fe uptake mechanism that can utilize deferiprone, desferal and many other chelators such as those disclosed in the prior art cited above (Stintzi, A., C. Barnes, J. Xu, K. N. Raymond. 2000). Pathogenic yeasts and other fungi can also utilize a variety of chelators as have been disclosed in the prior art cited above (Howard, D. H. 1999).
Chelating compositions comprised of a metal binding aspect affixed to an insoluble supporting carrier material have been disclosed previously. The compositions disclosed in U.S. Pat. No. 4,530,963, for example, relate to affixing known metal chelating molecules such as deferoxamine or catechol to an insoluble support material so as to provide an insoluble chelating composition. Such insoluble compositions enable the physical contact and removal of the composition with/from an aqueous medium to be treated. Such previously disclosed chelators are, however, inappropriate for treatment within an animal, including a human, due to their insoluble form. Insoluble compositions would not be suitable for administration into the body, for example into the blood stream.
Bacterial adhesion and biofilm formation are now recognized to be important cellular activities for bacterial and fungal pathogens during disease development (Hentzer M., M. Givskov, 2003). Typical iron chelators, such as desferrioxamine, have been shown to increase twitching motility and restrict biofilm formation in the laboratory (Singh et al, 2002). Appropriate restriction of iron supply during the early stages of bacterial or fungal disease may interfere with a pathogenic cell's activity of establishing a biofilm for example on an epithelial surface of the respiratory or urogenital tracts or on indwelling medical devices such as a urinary catheter. Iron chelators as disclosed in the prior art may suffer the same limitations for use in interfering with the activity of microbial adhesion for pathogens for those pathogens that can utilize these chelators or otherwise obtain iron from these.
There is therefore a need for iron chelating compounds that may be employed for sequestering iron. There is also a need for iron chelating compounds that are not utilizable, or easily utilizable, by the intended target cells. There is a further need for chelating compositions incorporating, for example, the metal binding properties of low molecular weight chelators in a structure where these chelators are affixed to a carrier that results in these being of sufficiently high molecular size so as not to be taken up into cells or be otherwise accessed for their iron by a cell.